Vector for integration site independent gene expression in mammalian host cells

ABSTRACT

Vectors for the integration of a gene into the genetic material of a mammalian host cell such that the gene may be expressed by the host cell comprise a promoter and the gene and include a dominant activator sequence capable of eliciting host cell-type restricted, integration site independent, copy number dependent expression of the gene.

This application is a continuation of application Ser. No. 07/920,536,filed Jul. 28, 1992, which is a continuation of Ser. No. 07/346,996,filed May 11, 1989, which was filed under 35 U.S.C. 371 as the nationalstage of the PCT/GB88/00655, filed Aug. 8, 1988.

FIELD OF THE INVENTION

The present invention relates to recombinant DNA technology and inparticular to a vector useful for transfecting mammalian cells in vivoand in vitro to obtain expression of a desired structural gene. Theinvention relates also to the use of such sequences in gene therapy andheterologous gene expression.

BACKGROUND TO THE INVENTION

There is a continuing need for improved expression vectors exhibitinghigh levels of expression. In particular expression vectors for use inmammalian cell lines are of increasing importance both for theindustrial production of desired polypeptides and for the development oftherapies for genetic disorders.

There are many known examples of characterized structural genes, whichtogether with appropriate control sequences may be inserted intosuitable vectors and used to transform host cells. A significant problemwith the integration of such a structural gene and control regions intothe genome of a mammalian cell is that expression has been shown to behighly dependent upon the position of the inserted sequence in thegenome. This results in a wide variation in the expression level andonly very rarely in a high expression level. The problem of integrationsite dependence is solved by the present invention and arises from thediscovery of sequences referred to herein as "dominant activatorsequences" (or "dominant control regions" (DCRs)) which have theproperty of conferring a cell-type restricted, integration siteindependent, copy number dependent expression characteristic of a linkedgene system.

The human β-like globin genes are a cluster of five genes in the order5'-ε-^(G) γ-^(A) γ-δβ-3' comprising approximately 60 kb of DNA on theshort arm of chromosome 11. The different genes are expressed in adevelopmentally and tissue-specific manner, i.e. the embryonic ε-gene isexpressed in the yolk sac, the fetal ^(G) γ and ^(A) γ genes, primarilyin the fetal liver, and the adult δ- and β-genes primarily in bonemarrow (for a review, see Maniatis et al., Ann. Rev. Genet., (1981) 14,145-178). Mutations in this gene family constitute the most widespreadfamily of genetic diseases and a large number of these have beencharacterized, ranging from simple amino acid changes by point mutationsto complete deletions of the locus (for a review, see Collins et al.,Prog. Nucl. Acid. Res. Mol. Biol., (1984) 31, 315-462). These often leadto severe clinical problems and early death. Conventional treatment ofthese diseases (transfusions) are costly, risky and, in many cases,inadequate. The present invention provides a therapy for such disordersfor example by gene therapy (Hock et al., Nature, (1986), 320, 275-277).

The DNA sequences which regulate the human β-globin gene are locatedboth 5' and 3' to the translation initiation site (Wright et al., Cell,(1984), 38, 265-273; Charnay et al., Cell, (1984), 38, 251-263). Usingmurine erythroleukemia cells (MEL) and K562 cells at least four separateregulatory elements required for the appropriate expression of the humanβ-globin gene have been identified; a positively acting globin specificpromoter element and a putatively negative regulatory promoter elementand two downstream regulatory sequences (enhancers), one located in thegene and one approximately 800 bp downstream from the gene (Antoniou etal., EMBO J., (1988), 7(2), 377-384). A similar enhancer has also beenidentified downstream of the chicken β-globin gene using culturedchicken erythroid cells (Hesse et al., PNAS USA, (1986), 83, 4312-4316,Choi et al., Nature, (1986), 323, 731-734). The downstream enhancer hasbeen shown to be a developmental stage specific enhancer usingtransgenic mice (Kollias et al., Cell, (1986), 46, 89-94 and NAR (1987),15, 5739-5747; Berringer et al., PNAS USA, (1987), 84, 7056-7060. All ofthese results therefore indicate that multiple development specificcontrol regions of the β-globin gene are immediately 5', inside and 3'to the β-globin gene.

However, when a β-globin gene containing all of these control regions isintroduced in transgenic mice, the gene is not expressed at the samelevel as the mouse β-globin gene and exhibits integration site positioneffects. This is characterized by a highly variable expression of thetransgene that is not correlated with the copy number of the injectedgene in the mouse genome. The same phenomenon has been observed inalmost all the genes that have been studied in transgenic mice (Palmiteret al., Ann. Rev. Genet., (1986), 20, 465-499). Moreover, the level ofexpression of each injected gene in the case of β-globin is, at best, anorder of magnitude below that of the enogenous mouse gene (Magram etal., Nature, (1985), 315, 338-340; Townes et al., EMBO J., (1985), 4,1715-1723; Kollias et al., Cell, (1986), 46, 89-94). A similar problemis observed when the β-globin or other genes are introduced intocultured cells by transfection or retroviral infection. This poses a bigproblem when considering gene therapy by gene addition in stem cells. Itis also a major problem for the expression of recombinant DNA productsfrom tissue cells. Extensive screening for highly producing clones isnecessary to identify cell-lines in which the vector is optimallyexpressed and selection for vector amplification is generally requiredto achieve expression levels comparable to those of the naturallyoccurring genes, such as for example β-globin genes in erythroleukemiccell-lines.

The study of the β-globin system has been assisted by analysis ofhemoglobinpathies such as thalassemias (van der Ploeg et al., Nature,(1980), 283, 637-642; Curtin et al., In: Hemoglobin Switching: FifthConference on Hemoglobin Switching, Washington Ed. G.Stamatoyannopoulos, Alan R. Liss, Inc., New York 1987). The Dutchthalassemia is hetrozygous for a large deletion which removes 100 kbupstream of the β-globin gene, but leaves the β-globin gene, includingall of the control regions described above intact (Kioussis et al.,Nature, (1983), 306, 662-666; Wright et al., Cell, (1984), 38, 265-273;Taramelli et al., NAR, (1986), 14, 7017-7029). Since the patient isheterozygous and transcribes the normal locus in the same nucleus, itindicates that some control mechanism may be overriding the functioningof the control sequences immediately surrounding the genes in the mutantlocus. In the case of the Dutch βthalassemia there are two possibleexplanations the observed effects; either a cis acting positive elementhas been removed or there has been insertion of a negative-actingelement in an inactive chromatin configuration and behaves like aclassical position effect (Koussis et al., Nature, (1983), 306,662-666). In a chromosome, the genetic material is packaged into aDNA/protein complex called chromatin which has the effect of limitingthe availability of DNA for functional purposes. It has been establishedthat many gene systems (including the β-globin system) possess so-calledDNase hypersensitive sites. Such sites represent putative regulatoryregions, where the normal chromatin structure is altered to allowinteraction with trans-acting regulatory regions.

Regions upstream from the epsilon-globin gene and downstream from theβ-globin gene which contain a number of "super" hypersensitive siteshave been identified. These sites are more sensitive to DNase Idigestion in nuclei than the sites found in and around the individualgenes when they are expressed. (Tuan et al. PNAS USA, (1985), 32,6384-6388; Groudine et al., PNAS USA, (1983), 80, 7551-7555. Inaddition, they are erythroid cell specific and they are present when anyone of the globin genes is expressed.

Tuan et al. describe the broad mapping of four major DNase Ihypersensitive sites in the 5' boundary area of the "β-like" globingene. The authors note that certain sequence features of these sites arealso found in many transcriptional enhancers and suggest that the sitesmight also possess enhancer functions and be recognized by erythroidspecific cellular factors.

The present invention arose from the discovery that the completeβ-globin locus with intact 5' and 3' boundary regions does not exhibitan integration site position dependence. The regions of the locusresponsible for this significant characteristic have been determined andshown to correspond to the DNase I super hypersensitive sites. Thesedominant activator regions are quite distinct from enhancers, exhibitingproperties such as integration site independence not exhibited by theknown enhancers. The dominant activator sequence used in conjunctionwith the known promoter/enhancer elements reconstitute full expressionof the natural gene. The connection made as part of the presentinvention between DNase I super hypersensitive sites and dominantactivator regions allows the invention to be extended to gene systemsother than the β-globin gene system. It has been shown, for example,that a human β-globin gene can be functionally expressed in transgenicmice in an integration site independent manner.

Human T-lymphocytes (T-cells) are produced in bone marrow and mature inthe thymus where they develop their immunological characteristic ofresponding to foreign antigens in the body. T-cells are produced in ahighly tissue specific manner. It is recognized that T-cells carryspecific cellular markers (Bernard et al. "Leukocyte Typing", Ed.Bernard et al., p 41, Springer Verlag, Berlin and New York, 1984). Onesuch marker is the E-rosette receptor, known by the designation CD2. Thestructure of the CD2 marker and the CD2 genes has been the subject ofconsiderable study (Brown et al., Met. in Enzyme., (1987), 150, 536-547and Lang et al., EMBO J, (1988), 7(6), 1675-1682).

In the latter paper, published after the priority date of the presentinvention, the property of a large fragment including the CD2 gene toexhibit copy number dependence is noted and a suggestion is made thatsuch a fragment contains a locus forming sequence analogous to thosefound in β-globin. DNase super hypersensitive sites have now beenidentified in the fragment and shown to be dominant activator sequencesof the present invention.

Finally, the present invention is applicable to the production oftransgenic animals and the techniques for producing such are now widelyknown. For a review, see Jaenisch, Science, (1988), 240, 1468-1474.

The present invention provides a solution to the problem of integrationsite dependence of expression making possible the insertion offunctionally active gene systems into mammalian genomes both in in vitroand in vivo. Specific sequences providing advantageous increases intranscription levels have also been identified.

SUMMARY OF THE INVENTION

According to the present invention there is provided a vector for theintegration of a gene into the genetic material of a mammalian host cellsuch that the gene may be expressed by the host cell, the vectorcomprising a promoter and the gene characterized in that the vectorincludes a dominant activator sequence capable of eliciting hostcell-type restricted, integration site independent, copy numberdependent expression of the gene.

As used herein the term "dominant activator sequence" means a sequenceof DNA capable of conferring upon a linked gene expression system theproperty of host cell-type restricted, integration site independent,copy number dependent, expression when integrated into the genome of ahost cell compatible with the dominant activator sequence. Such adominant activator sequence retains this property when fullyreconstituted within the chromosome of the host cell. The ability todirect efficient host cell-type restricted expression is retained evenwhen fully reformed in a heterologous background such as a differentpart of the homologous chromosome (e.g., chromosome 11 for β-globin) orindeed a different chromosome altogether.

It is hypothesized that the dominant activator sequences of theinvention may open the chromatin structure of the DNA, making it moreaccessible and thus may act as a locus organizer. The dominant activatorsequence may be a single contiguous sequence corresponding to, orderived from a naturally occurring gene system or may consist of two ormore such sequences linked together with or without interveningpolynucleotides.

The dominant activator sequence may be derived by recombinant DNAtechniques from a naturally gene system or may correspond to a naturallyoccurring gene system in the sense of being manufactured using knowntechniques of polynucleotide synthesis from sequence data relating to anaturally occurring gene system. Alterations of the sequence may be madewhich do not alter the function of the dominant activator sequence.

Preferably, the naturally occurring gene system from which the dominantactivator sequence is derived or with which it corresponds is a systemwhich exhibits a highly host cell-type restricted expressioncharacteristic preferably at a high level. Specific examples of suchsystems are the hemoglobin systems such as β-globin system andlymphocyte systems such as the CD2 system.

The dominant activator sequence may consist of, be derived from, orcorrespond to one or more DNase I super hypersensitive site, preferablyof any gene system capable of cell specific expression. Other sequencesmight however exhibit the functional characteristics of a dominantactivator sequence. Where the naturally occurring dominant activatorsequence comprises two or more subsequences separated by an interveningpolynucleotide sequence or sequences the dominant activator sequence maycomprise two or more of the subsequences linked in the absence of all ora part of one or more of the intervening sequences. Thus, if thedominant activator sequence of a naturally occurring gene locuscomprises two or more discrete subsequences separated by intervening nonfunctional sequences, (for example, two or more super hypersensitivesites) the vector of the invention may comprise a dominant activatorsequence comprising two or more of the subsequences linked together withall or part of the intervening sequences removed.

The term "vector" as used herein connotes in its broadest sense anyrecombinant DNA material capable of transferring DNA from one cell toanother.

The vector may be a single piece of DNA in linear or circular form, andmay, in addition to the essential functional elements of the invention,include such other sequences as are necessary for particularapplications. For example, the vector may contain additional featuressuch as a selectable marker gene or genes, and/or features which assisttranslation or other aspects of the production of a cloned product. Thevector suitable for integration consists of an isolated DNA sequencecomprising a dominant activator sequence and an independent structuralgene expression system.

The DNA sequence is not linked at either end to other substantial DNAsequences. The isolated DNA sequence may however be provided withlinkers for ligation into a vector for replication purposes or may beprovided with sequences at one or both ends to assist integration into agenome.

The vector defines a "mini locus" which can be integrated into amammalian host cell, where it is capable of reconstructing itself in ahost cell-type restricted, integration site independent, copy numberdependent manner.

The invention also provides a transfer vector, suitably in the form of aplasmid, comprising a dominant activator sequence. Such vectors areuseful in the construction of vector for integration.

The term "gene" as used herein connotes a DNA sequence, preferably astructural gene encoding a polypeptide. The polypeptide may be acommercially useful polypeptide, such as a pharmaceutical and may beentirely heterologous to the host cell. Alternatively the gene mayencode a polypeptide which is deficient absent or mutated in the hostcell.

The mammalian host cell may be any mammalian host cell susceptible touptake of the vector of the invention. The vector DNA may be transferredto the mammalian host cell by transfection, infection, microinjection,cell fusion, or protoplast fusion.

The host cell may be a cell of a living human or animal. In particular,the host cell may be a cell of a transgenic animal such as a mouse. Thehost cell may be a human stem cell such as bone marrow cell. The hostcell may be derived from tissue in which the dominant activator sequenceis functional, such as erythroid cells in the case of the β-globinlocus.

The promoter may be any promoter capable of functioning in the host celland may be for example a mammalian or viral promoter. Optionally, thepromoter may be homologous with the gene locus of the dominant activatorsequence (for example the β-globin promoter) and may be present intandem with another promoter (for example the β-globin promoter and aviral TK or SV40 promoter) and may include one or more enhancerelements.

In one embodiment of the invention to be described below, the dominantactivator sequence is derived from the β-globin gene locus. As discussedabove, the β-globin locus contains a number of DNase I superhypersensitive sites which constitute the dominant activator regions(see Tuan et al. loc cit). Preferably dominant activator sequencecontains one or more of the DNase I super hypersensitive sitesidentified within the β-globin locus. Preferably these are from the 5'boundary of the locus, optionally with the 3' boundary sequences. Thedominant activator sequence is within a fragment of 21 kb from -1 kbClal to -22 kb BglII immediately upstream of the epsilon-globin gene inthe β-globin locus. This region contains four DNase I superhypersensitive sites with intervening polynucleotides (five distinctsites of which two are very close together). Preferably some or all theintervening nucleotides are removed using known techniques such asdigestion with exonuclease.

A reduced form of the β-globin locus dominant activator sequence hasbeen produced which exhibits a significantly increased level ofexpression of a linked gene expression system. This was produced byligating the following four fragments:

2.1kb XbaI-XbaI

1.9kb HindIII-HindIII

1.5kb KpnI-BglII

1.1kb partial SacI fragment

This dominant activator sequence, referred to herein as a "micro locus"is a 6.5 kb fragment which may be used as a "cassette" to activate aspecific gene expression system.

In a second embodiment of the invention to be described below, thedominant activator sequence is derived from the CD2 gene locus. The CD2gene locus contains three super hypersensitive sites; one at the 5'boundary of the locus and two at the 3' boundary of the locus.Preferably the dominant activator contains one or more of the DNase Isuper hypersensitive sites within the CD2 locus. Most preferably, thedominant activator sequence contains both the super hypersensitive sitesfrom the 3' boundary of the locus, optionally with all or a part of anyintervening sequence deleted. The dominant activator sequence iscontained within a 5.5 kb BamHI to XbaI fragment 3' to the CD2 gene. Ina further aspect of the invention there is provided a method ofproducing a polypeptide comprising culturing a host cell transformedwith a vector of the invention.

The method may be applied in vitro to produce a desired polypeptide. Inaddition, the method may be applied in vivo to produce a polypeptidehaving no therapeutic value to the animal. Such a method of producing apolypeptide is not to be considered as a method of treating the human oranimal body.

In a further aspect of the invention the vector may be used in a methodof treatment of the human or animal body by replacing or supplementing adefective mammalian gene.

Many diseases of the human or animal body result from deficiencies inthe production of certain gene products. The characteristic features ofthe vectors of this invention make them amply suited to the treatment ofdeficiencies by gene therapy in vivo.

A method of gene therapy is provided comprising removing stem cells fromthe body of a human or an animal, killing stem cells remaining in thebody, transforming the removed stem cells with a vector of the inventioncontaining a gene deficient, or absent, in the human or animal body, andreplacing the transformed stem cells in the human or animal body. Thismethod can be used to replace or supplement a gene deficient in a humanor animal. For example, an individual suffering from a hemoglobinopathysuch as β-thalassemia could be treated to insert an active highlyexpressed β-globin gene locus to make up the deficient β-hemoglobin.Alternatively a deficiency in the immune system could be treated with aCD2 locus vector. This method could be used to treat deficiencies inadenosine deaminase (ADA syndrome) or severe combined immune deficiencysyndrome (SCID syndrome).

Bone marrow is a suitable source of stem cells and is advantageous inthat it contains the precursors of both lymphocytes and erythroid cells.Alternatively other tissue could be removed from the body, transfectedor otherwise provided with a vector of the invention and implanted backinto the body.

The invention is now described by way of example with reference to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A-D) shows the mapping of DNase I super hypersensitive site 5'to the epsilon globin gene. Panels A to C are representations ofnitrocellulose filters illustrating the mapping experiment and Panel Dshows a restriction map of the β-globin locus upstream of the epsilonglobin gene for the three restriction enzymes used in these experiments.Probe locations and super hypersensitive sites are marked.

FIG. 2 (A-E) shows the corresponding experiment to that in FIG. 1conducted to map the super hypersensitive sites 3' of the β-globin gene.

FIG. 3 shows the construction of a human 3-globin "mini" locus.

FIG. 4 (A-C) shows a structural analysis of the human β-globin transgene(FIG. 4 comprises 4A, 4B, 4C).

FIG. 5 (A-D) shows an expression analysis of the human β-globintransgene in mouse (FIG. 5 comprises 5A, 5B, 5C, 5D).

FIG. 6 shows the production of human β-globin DNA in various mousetissues (ys=yolk sac, lv=Fetal liver and br=brain)

FIG. 7 (A-F) shows the reformation of DNase I hypersensitive sites onthe transgenic mini β-globin locus.

FIG. 8 shows the construction of plasmids GSE1364, 1365 and 1366.

FIG. 9 shows the construction of plasmids GSE1359, 1400, 1401 and 1357.

FIG. 10 shows human β-globin expression.

FIG. 11 (A-D) shows the mapping of CD2 hypersensitive sites.

FIG. 12 shows a Thy-1 CD2 construct.

FIG. 13 shows the distribution of Thy-1 in the thymus and brain oftransgenic mice.

FIG. 14 shows the construction of a β-globin CD2 construct.

FIG. 15 shows the genomic DNA of transgenic mice carrying the constructof FIG. 14.

EXAMPLE 1

Experiments were conducted to establish whether dominant control regionsare present in the flanking regions of the β-globin locus by includingsuch regions in a β-globin construct. If such sequences were insensitiveto integration site position effects, it would be expected: (1) that thelevel of expression of the construct is directly related to its copynumber in the transgenic mice, (2) that the level of expression per genecopy is the same in each mouse and (3) possibly that the introduced geneis expressed at a level comparable to that of the mouse genes. Prior totesting the 5' and 3' sequences, it was important to carefully map thepositions of the hypersensitive sites since their exact position,especially at the 3' side, has not been published in detail (Tuan etal., 1985).

Mapping DNase hypersensitive sites flanking the β-globin locus

Very limited DNase I digestion of nuclei of erythroid cells revealssites extremely sensitive to DNase upstream of the epsilon-globin geneand downstream of the β-globin gene (Tuan et al., 1985). We fine mappedthe DNase I hypersensitive sites relative to restriction sites mapped inour cosmids (Taramelli et al., 1986). FIG. 1 shows a time course ofDNaseI digestion on nuclei of HEL (Martin and Papayannopoulou, 1982) andPUTKO (Klein et al., 1980) cells recut with various restriction enzymes,Southern blotted and hybridized with a number of probes upstream of theepsilon-globin gene (FIG. 1D). Four hypersensitive sites are present at18 kb (No. 1), 15 kb (No. 2), 12.0 and 11.5kb (No. 3A, B) and 5.4 kb(No. 4) upstream of the epsilon-globi gene. Nos. 1, 3A and 4 sites arestrong hypersensitive sites as judged by their early appearance duringthe time course of digestion. Nos. 2 and 3b sites are weaker sites andappear later during the time course of digestion. In FIG. 1, theexperimental details were as follows:

Panel A

Nuclei of HEL cells were incubated with DNase (60 μg/ml) at 37° C. andaliquots removed from the reaction at the times indicated (in minutes)above each track. Aliquots of approximately 10⁷ HEL nuclei wereincubated in the absence of DNase I (0 enz.) or presence of 30 units ofAlul or 60 units of Hinfl for 15 min. at 37° C. DNA was purified afterproteinase K digestion, recut with Asp718, fractioned on a 0.6% aragosegel transferred to nitrocellulose and probed with an epsilon-globin geneprobe (Bam-Eco 1.4kb).

Panel B

The DNase I digested samples of Panel A were recut with BglII and probedwith a 3.3 kb EcoRI fragment which in addition to 12.0 and 5.8 kb bandsalso detects a cross hybridizing band of 6.8 kb after washing filters ata stringency of 0.3×SSC at 65° C., which is not observed on washing at0.1×SSC at 65° C. (not shown).

Panel C

Nuclei of PUTKO cells were digested with DNaseI (20 μg/ml) at 37° C. andsamples removed from the reaction after 1,2,4 and 8 minutes. An aliquotof approximately 10⁷ nuclei was incubated for 8 minutes at 37° C. in theabsence of DNase I (lane 0 enz.). After recutting with BamHI, gelelectrophoresis, and transfer to nitrocellulose, the filter washybridized with a 0.46 kb EcoRI-BglII probe and washed to a finalstringency of 0.3×SSC at 65° C.

Panel D

Shows a restriction map of the β-globin locus upstream of theepsilon-globin gene for the three restriction enzymes used in theseexperiments. Location of probes and deduced positions of DNaseIhypersensitive sites in HEL and PUTKO chromatin are marked.

The exact location of the DNaseI hypersensitive site 3' of the adultβ-globin gene was determined using two single copy DNA probes andseveral restriction enzyme digests of DNase I digested HEL nuclei. Thedata summarized in FIG. 2 (A-D) show that there is a single DNaseIhypersensitive site between the 2.3 kb BglII fragment and the 2.4 kbHindIII fragment approximately 20 kb 3' of the adult β-globin gene (FIG.2E). (Similar results were obtained with DNaseI digested PUTKO nuclei(data not shown)).

In FIG. 2 the experimental details were as follows:

Panels A to D show DNA from DNaseI digested nuclei of HEL cells recutwith the indicated restriction enzymes and probed with either a 1.15 kbEcoRI or a 1.1 kb HindIII-BamHI fragment. Both DNA probes contain somehuman repetitive DNA which necessitated the use of 10μg sheared humanDNA per ml in prehybridization and hybridization buffers. Filters werewashed to a final stringency of 0.3×SSC at 65° C.

Panel E shows a restriction map of the β-globin locus 3' of the β-globingene. The position of the two probes used and the 3' HSS site aremarked. The position of HSS 5 was confirmed by probing HindIII and BglIIrecut DNA samples with the 1.15 EcoRI probe (data not shown).

Construction of the β-globin "mini" locus

The globin "mini" locus (FIG. 3) was constructed from three regions (see"Materials and Methods" below for the precise description); a 21 kbregion immediately upstream of the epsilon-globin gene (-1 kb ClaI to-22 kb BgII) containing all four hypersensitive sites (FIG. 1), a 4.7 kb(BglII) fragment containing the β-globin gene and all the knownregulatory sequences immediately in and around the gene and a 12 kbregion downstrean from the β-globin gene (+12 kb KpnI to +24 kb BamHI)containing the downstream hypersensitive site (FIG. 2). These regionscontained or were provided with unique linkers to create a series ofunique restriction sites (SalI, XhoI. ClaI. KmnI, PvuI) and cloned intothe cosmid pTCF (Grosveld et al., 1982). The entire 38 kb insert wasexcised from the cosmid as a SaII fragment, purified from low meltingagarose and injected into fertilized mouse eggs (Kollias et al., 1986).

Referring to FIG. 3, the human β-globin locus is illustrated on top andthe sizes are shown in kilobases (kb). The second line shows theβ-globin "mini" locus, with a number of unique restriction sites; theconnecting lines indicate the original position of the fragments in theβ-globin locus. The restriction enzyme fragments in the "mini" locus forEcoRI. BamHI and HindIII are shown in kb. The hybridization probes forthe 5' flanking region (EcoBgl 0.46), the β-globin gene (BamEco 0.7 IVSII) and the 3' flanking region (Eco 1.15) are shown at the bottom.

Transsenic mice containing the β-globin "mini" locus

Initial injection of mouse eggs was carried out with a DNA concentrationof 2μg/ml, the eggs were transplanted back into the mice, but nooffspring were obtained (for unknown reasons). Several series ofinjections were then carried out with a DNA concentration of 0.5-1 μg/mland fetuses were collected after either 12.5 or 13.5 days of gestation.DNA was prepared from individual placentas, Southern blotted andhybridized with a human β-globin probe to determine the number oftransgenic mice (as described by Kollias et al., 1986). The transgenicDNAs were subsequently digested with EcoRi, Southern blotted andhybridized with a human β-globin probe and a mouse Thy-1 probe (singlecopy) to determine the number of genes integrated in each mouse. (FIG.4A and Table 1--see page 29). These values varied from a single insert(mouse 33 and 38) up to a hundred copies per cell (mouse 21). It wasthen determined whether the inserted DNA was integrated intact orwhether deletions and/or rearrangements had taken place after injection.Total DNA was digested with BamHI or HindIII, Southern blotted andprobed for the presence of the complete construct. In particular a 0.46kb BglII probe (FIG.3) was used to detect the 15 kb BamHI fragment atthe 5' end of the construct (FIG. 4B) and a 1.15 kb EcoRi probe (FIG. 3)to detect the 14.6 kb BamHI or 18 kb HindIII fragment at the 3'end ofthe construct (FIG. 4C). All but one of the transgenic mice appeared tohave intact "mini" β-globin loci which are integrated in tandem arrays(not shown) as judged by the presence of the correctly sized fragments.It should be noted however that the BamHI and HindIII sites at the 3'end of the locus (FIGS. 4 and 6) are very resistant to cleavage, eventhough the mixed-in control DNA has been fully digested (see Methods).This results in partial cleavage bands which are larger than fullydigested BamH (14.6 kb) and HindIII (18kb) fragments. Transgenic mouse38 is the only mouse that has not retained a full copy of the β-globin"mini" locus. The single copy present in this mouse has lost the BamHIand HindIII sites upstream of the β-globin gene, although the EcoRI siteis still present. This indicates that all the 5' flanking DNA includingthe whole hypersensitive site region has been deleted in this mouse.

In FIG. 4, the experimental details were as follows:

Panel A

Approximately 5gg of placental or yolk sac DNA was digested with EcoRI,Southern blotted after gel electrophoresis and hybridized to either thehuman β-globin Bam-Eco IVS II probe or a mouse Thy-1 probe. The gelswere quantitated by densitometer scanning of different exposures. TheDNA of mouse 17 was partially degraded, therefore, a lower M.W. Thy-1cross hybridizing band was used to quantitate this sample. The inset (ahigh exposure of the globin hybridization) shows the low copy signals.

Panel B

Placental or yolk sac DNA was digested with BamHI, Southern blotted andhybridized to a 5' flanking probe (Eco-Bgl 0.46, FIG. 3).

Panel C

Placental or yolk sac DNA was digested with BamHI or HindIII, Southernblotted and hybridized to a 3' flanking probe (Eco 1.15, FIG. 3).

In all panels a mixture of marker DNA was used from a DNA digested withBstEII or HindIII. The transgenic mice are indicated at the top, emptylanes contain DNA samples from non-transgenic mice. Sizes are inkilobases (kb). Some of the DNA samples are partially digested, althoughthe mixed-in DNA control was completely digested (not shown). Thisresult in bands which are larger than the 14.6 kb BamHI(14.6kb+3.2kb+3.3kb) or the 18kb HindIII fragments (18kb+2.4kb+3.4kb) bycleavage at the next BamHI or HindIII site in the tandem arrays.

The human β-globin "mini" locus is fully active

To measure the activity of the human β-globin gene, RNA was preparedfrom all the transgenic and non-transgenic mice (liver, blood and brain)and analyzed by S1 nuclease protection analysis. FIG. 5A shows theanalysis of liver RNA with a 5' human β-globin, while FIG. 5B shows ananalysis of the 3' end of the human β-globin RNA in the presence of a 3'mouse β-globin and mouse α-globin probes as internal controls. Theseresults show that the human β-globin gene is correctly initiated andpolyadenylated and that the levels of RNA are proportional to the numberof copies of the "mini" locus in each mouse (except 24 and 38, seebelow). Moreover, no expression is found in non-erythroid tissue (notshown). The level of expression is clearly very high when the ratio ofthe human β- to mouse β-globin RNA levels (Table 1 see page 29) iscompared to that of the Hull cells control (Zavodny et al., 1983). Thelatter cell line is a mouse erythroleukemia (MEL) cell line containingthe human β-globin locus on a human X-11 hybrid chromosome. Tocarefuilly quantitate the levels of expression in these mice, RNA levelsin mice 33 and 12 which have one and two intact copies of the β-globinlocus were analyzed, and the two exceptions mice 24 and 38 with mouseand human β-globin probes of equal specific activity. FIG. 5C shows thatthe signal of the human β-globin gene is approximately half that of themouse β-globin diploid in mouse 33 and equal in mouse 12. This isconfirmed at the protein level when the red cells of mouse 12 and anage-matched control are lysed and the proteins run on a Triton-acid ureagel (Alter et al., 1980). It is clear that the transgenic mouse 12expresses equal levels of mouse and human β-globin proteins (FIG. 5D).From all these data it was concluded that each human β-globin gene inthese mice is fully active, i.e. at a level equivalent to that of themouse β-globin genes, while the expression in mice 24 and 38 issuppressed. A peculiar situation is found in mice 21 and 17. The mouseβ-globin RNA levels are very low in these mice, presumably as a resultof the very high copy number of the human β-globin gene and, as aconsequence, very high mRNA (and protein) levels. The high copy numberpossibly results in a competition for transcription factors (mouseβ-globin RNA is decreased more than mouse α-globin RNA, FIG. 5B), whilethe high levels of β-globin protein definitely result in an anemia(α-thalassemia). Mice 21 and 17 had small colorless livers consistentwith the destruction of mature red cells and a concomitant loss of mRNA.

Two mice (38 and 24) form an exception because they express relativelylower amounts of human β-globin (FIG. 5A, B, C, Table I see page 29).The situation in mouse 38 is explained by the fact that this is the onlytransgenic mouse which has a deletion of the 5'end of the locus (FIG.4). As a consequence, the human β-globin gene is still expressed, but ina position dependent manner and at low levels, just as found when smallβ-globin fragments are introduced in mice (Kollias et al., 1986; Magramet al., 1985; Townes et al., 1985).

Mouse 24 has no apparent defect in the locus and the possibility wasinvestigated that this mouse is a true exception or whether it is eithera chimeric mouse or has the β-globin locus integrated on an inactiveX-chromosome. DNA was therefore isolated from the yolk sac, brain andfetal liver (the "erythroid" tissue) and hybridized the EcoRI digestedDNAs on Southern blots with a human β-globin probe and a murine Thy-1probe to detect the β-globin transgene versus an endogenous single copygene. FIG. 6 clearly shows that 10 μg of fetal liver DNA contains evenless than half of the globin signal that 5 μg of yolk sac DNA, i.e.,much less than 25% of the liver cells contain the β-globin gene. This istherefore a chimeric mouse, in which the β-globin mini locus isunder-represented in the erythroid tissue (fetal liver) which explainsthe lower levels of human β-globin mRNA found in these cells. It may beconcluded from this that provided the locus has not been affected, thereis a complete correlation between expression levels and DNA copy numbersand that each gene is fully expressed in every transgenic mouse. In FIG.5, the experimental details were as follows:

Panel A:

5 μg of RNA from 12.5 or 13.5 day livers from different mice (numbers ontop) was analyzed by S1 nuclease protection as described previously(Kollias et al., 1986). The protected fragment of the 5' human β-globinprobe (525bp AccI from an IVS I lacking β-globin gene) is 160nt (5'βglo). Input probe is shown as ip. The marker (m) is pBR322 DNA digestedwith Hinfi and the sizes are indicated in nucleotides.

Panel B 5μg of RNA was analyzed as in panel A, but using two probes; a3' probe of the human β-globin gene (760 bp EcoRI-MspI) resulting in a210 nt protected fragment (Hbeta) and a mouse α-globin probe (260 bpRamlH second exon probe), resulting in a 170 nt protected fragment (Malpha). The positive control is RNA from the hybrid MEL cell line Hull.Marker (m) is pBR322 ×Hinf.

Panel C 5 μg of RNA was analyzed as in panel B, but in addition, a mouseβ-maj-globin probe (a 730 bp second exon probe) was added resulting in a100 nt protected fragment (Mbeta). The probes were of equal specificactivity. The activity of the probes was assessed by quantitativeagarose, gel electrophoresis and autoradiography of the individualprobes before and after mixing for the S1 nuclease analysis (not shown).The positive control is Hull RNA (hu). Each probe is individually testedwith RNA from Hull (hu) or non transgenic mouse fetal liver RNA (ml).

Panel D

Samples of fetal liver (approximately 20μg) or human red cells werelysed in 40 μl of H₂ O and the globin proteins separated as described(Alter et al., 1980). The gel was stained to visualize the globinprotein chains. Mouse 9 is an age-matched non-transgenic (litter) mateof mouse 12. The positions of mouse α-, β- and human β-globin areindicated.

Mapping DNaseI hypersensitive sites in the transgenic β-globin locus

The DNaseI hypersensitive sites flanking the β-globin locus are found inall erythroid tissues studied regardless of which β-like globin isexpressed. To determine if the DNaseI hypersensitive sites are reformedin the mini β-locus of the transgenic mice expressing high levels ofhuman β-globin mRNA, a DNaseI fadeout was performed on a small sample ofthe liver and brain of mouse 21 (which has the highest copy number ofthe βgene mini locus). Limited DNaseI digestion was carried out in thepresence of carrier fetal liver or brain nuclei, purified nuclear DNAwas recut with BamHIl, fractioned, Southern blotted and fragmentsspanning the whole construct were used as hybridization probes. The 1.15EcoRI probe which was used to detect the 3' hypersensitive site in HELand PUTKO cells detects a 17.8 kb BamHI fragment which is due to partialdigestion of the BamHI site at the 3' end of the injected fragment. The17.8 kb BamHI fragment is completely insensitive to limited DNaseIdigestion in liver nuclei of mouse 21 (FIG. 7E). The same filterreprobed with a 600bp AccI 3' human β-globin probe shows that the 2.0 kbBamHI fragment encompassing the 5' end (to -1460bp) of the highlytranscribed human β-globin gene is slightly sensitive to DNaseIdigestion, revealing a DNaseI sub-band of approximately 1 kb (FIG. 7D),consistent with a DNaseI hypersensitive site within the β-globin genepromoter (Groudine et al., 1983). Reprobing the same filter with the0.46 EcoRI GblII probe (FIG. 1C) shows that the 15 kb BamHI fragmentencompassing hypersensitive sites 2, 3a, and 4 is exceedingly sensitiveto DNaseI digestion in fetal liver nuclei and sub-bands corresponding tocutting at sites 3 and 4 are clearly visible. The same 15 kb BamHIfragment of the transgene mini β locus is insensitive to DNaseIdigestion in brain nuclei of the same animal (not shown). The 3.3 kbBamHI fragment (containing hypersensitive site no. 1) of the transgenicmini locus is also very sensitive to DNaseI digestion in liver, but notbrain nuclei of mouse 21 (FIG. 7A, B).

In summary, the 5' flanking region is extremely sensitive to DNaseIdigestion and has retained the superhypersensitive sites in a tissuespecific fashion. Surprisingly, the 3' flanking region is insensitive toDNase digestion and the super hypersensitive site is not retained. Thenormal hypersensitivity in the promoter is present (Groudine et al.,1983).

In FIG. 7, the experimental details were as follows:

Nuclei of mouse 21 tissues were incubated with 5 μg DNase per ml for0,1,2,4 and 8 minutes at 37° C. (liver nuclei), or 0,1,2,5 and 10minutes (brain nuclei). Aliquots of nuclei were incubated for 8 or 10minutes in the absence of DNase (lanes 0 end.). Purified DNA was recutwith BamHI fractioned on a 0.6% agarose gel, transferred tonitrocellulose and probed with DNA fragments spanning the transgenichuman β-globin mini locus (Panels A-E).

The 14.0 kb BamHI fragment detected by probe D in panel E is a junctionfragment of the tandem array of the mini β-globin locus. The 17.8 kbband is the result of incomplete BamHI digestion.

Panel F shows a schematic representation of a repeat unit of the minilocus tandem array in mouse 21 on which DNase hypersensitive sites aremarked. Site 5 is not reformed in fetal liver nuclei (Panel F). DNaseIsub-bands corresponding to HSS 1 and 2 are not seen in DNaseI fadeoutexperiments, but the BamHI fragments in which they reside are sensitiveto DNaseI digestion in liver (Panels A and C but not brain (Panel B anddata not shown)).

                  TABLE 1                                                         ______________________________________                                        Levels of human β-globin RNA and DNA.sup.1                               Mouse No. β/α RNA.sup.2                                                                 β/Thy-1 DNA.sup.2                                                                   RNA/DNA                                       ______________________________________                                        12        1.0        1.0        1.0                                           17        42.04      47.2.sup.3 0.9                                           ______________________________________                                         .sup.2 The ratios were obtained by dividing the human globin RNA and DNA      signals by those obtained for the mouse globin RNA and Thy1 DNA,              respectively. Mouse 34 RNA was measured in only one experiment and not        included.                                                                     .sup.3 The DNA of mouse 17 was partially degraded (see FIG. 4). We            therefore used a low M.W. cross hybridizing band on the Thy1 blot to          quantitate the copy number (rather than the degraded 8kb EcoRI Thy1 band)     .sup.4 The ratio of human globin to mouse golobin RNA was not determined      (N.D.) accurately in this mouse, due to the very heavy high human to mous     RNA signals (see text).                                                       .sup.5 Mouse 24 is chimeric for the human globin gene (FIG. 6) and could      therefore not be quantitated.                                            

² The ratios were obtained by dividing the human β-globin RNA and DNAsignals by those obtained for the mouse α-globin RNA and Thy-1 DNA,respectively. Mouse 34 RNA was measured in only one experiment and notincluded.

³ The DNA of mouse 17 was partially degraded (see FIG. 4). We thereforeused a low M.W. cross hybridizing band on the Thy-1 blot to quantitatethe copy number (rather than the degraded 8kb EcoRI Thy-1 band).

⁴ The ratio of human β-globin to mouse globin RNA was not determined(N.D.) accurately in this mouse, due to the very heavy high human tomouse RNA signals (see text).

⁵ Mouse 24 is chimeric for the human β-globin gene (FIG. 6) and couldtherefore not be quantitated.

MATERIALS AND METHODS

Transzenic Mice

The 38 kb SalI fragment was purified from cosmid vector sequences by gelelectrophoresis and prepared for injection as described (Kollias et al.,1986; Brinster et al., 1985). Transgenic fetuses were identified bySouthern blot analysis (Southern, 1975) of placental DNA.

RNA analysis

RNA extraction of different tissues and S 1 nuclease protection analysiswere carried out as described previously (Kollias et al., 1986).

DNaseI sensitivity

DNase1 sensitivity assays were carried out on isolated nuclei asdescribed by Enver et al. (1984) except that 1 mM EGTA was included inall buffers until DNaseI digestion in buffer A plus 1 mM CaCl₂ wasperformed. In the case of transgenic liver and brain, four livers andbrains from age-matched normal fetuses were added as carriers to providesufficient material to carry out the analysis.

Construction of the β-globin mini locus

(1) A 12 kb BglII fragment (10-22kb 5' of β-globin) was cloned into theBamHI site of cosmid pTCF (Grosveld et al., 1982) without a ClaIsite→cosHG B12.

(2) cosHG B12 was cleaved with Asp718 (13.5 kb 5' of epsilon-globin) anda unique XhoI linker was inserted→cos HG B12X.

(3) cosHG 14.2 (Taramelli et al., 1986) containing the entireepsilon-globin upstream region was also cleaved with DpnI and XhoIlinker inserted and packaged→cos HG 14.2X.

(4) A ClaI linker was inserted in the XbaI site of pUC18 →pUC 18C.

(5) The 4.7 kb BglII fragment containing the β-globin gene was insertedin the BamHI site of pUC18C→pUC18C-BG.

(6) A 12 kb HpaI-BamHI fragment (12-24 kb 3' of β-globin) was clonedinto HpaI-BamHI of cosmid pTCF lacking a ClaI site→cos HG HB 12.

(7) The final cosmid construct was put together as a four fragmentligation and packaging, a 16.5 kb PvuI-XhoI fragment from cos HG B12Xcontaining the cosmid and part of the upstream sequences, an 11 kbXhoI-ClaI fragment from cos HG 14.2X containing the remainder of theupstream sequences, a 4.7 kb ClaI-KpnI fragment from pUC18C-BGcontaining the β-globin gene and a 19 kb KpnI-HpaI fragment from cos HGHB12 containing the 3' flanking region and the second cosmid forpackaging purposes→cos HG "mini" locus (FIG. 3).

DISCUSSION OF EXAMPLE 1

Position effect independent expression

The introduction of the human β-globin gene flanked by the upstream anddownstream regions in transgenic mice has resulted in an expressionpattern that is position independent and directly related to copynumber, with levels of human β-globin mRNA as high mouse β-globin mRNA.This is different from that observed with previous transgenic mice usingthe β-globin gene alone (Magram et al., 1985; Chada et al., 1985; Towneset al., 1985; Kollias et al., 1986,1987), or other genes such as theimmunoglobulin genes (Grosschedl et al., 1984), albumin genes (Pinkertet al., 1987), Thy-1 gene (Kollias et al., 1987) AFP gene (Hammer etal., 1987) and many others. As a result of position effects in alltransgenic mice experiments to date, the level of transgenic expressionhas been suboptimal and has varied between different mice, i.e. therehas been no strict correlation between the copy number of the transgeneand the level of expression. Therefore, it has been very difficult toquantitate any experiments accurately and, as a result, it has beenimpossible, for example, to examine the co-operation between regulatoryelements in any gene. The results described in this example indicatethat this problem can be overcome for the β-globin gene. It is clearthat the expression levels found in mice containing the intact minilocus is directly proportional to the number of genes that have beenincorporated (Table I). In addition, the expression level per humanβ-globin gene in all of those mice is very similar to that observed forthe endogenous mouse β-globin gene (FIG. 5). It is therefore concludedthat the β-globin locus flanking sequences confer position independentexpression on the β-globin gene which itself is erythroid specific. Inaddition it is concluded that all of the regulatory sequences involvedin β-globin gene expression are included in this construct.

Gene Therapy

Perhaps the most interesting application for the properties of thedominant control region(s) is that they might allow completely regulatedexpression of the β-globin (and possibly other genes) in retroviralvector or transfection systems. Gene expression in such systems ispresently position dependent and inefficient. In particular, retroviralvector systems which are presently the only efficient way to transfergenes into hematopoietic (or other) stem cells, are very sensitive tothe integration site of the retroviral vector (Cone et al., 1987).Inclusion of the globin dominant control regions may solve this problemand allow the efficient transfer of a fully active, single copy humanglobin gene to hematopoietic stem cells. This would form the basis forgene therapy by gene addition in the case of thalassemias.

EXAMPLE 2

A β-globin "micro locus" was constructed including all four of the five5' DNaseI superhypersensitive sites (HSS). The method described below isgeneral and the micro locus produced shows an advantageous increase inexpression levels. The method described below may therefore be appliedto the construction of other micro loci from dominant activator regionsof other genes.

Methods for the construction of a β-globin micro locus

To facilitate DNA manipulations required to place all four 5' DNaseIhypersensitive sites and the human β-globin gene in a vector containingthe tk neo gene, a series of polylinker vectors were constructed byinserting oligonucleotides between the AatII and PvuII sites of theplasmid vector pUC 18 to generate plasmids GSE 1364, GSE 1365, and GSE1366 (see FIG. 8). The required DNA fragments were cloned into theunique restriction sites created in the different polylinker vectors. Inparticular:

1. A 2.1 kb XbaI--XbaI fragment encompassing DNaseI HS 1 was cloned intothe XbaI site of GSE 1364,

2. A 1.9 HindIII--HindIII fragment was cloned into the HindIII site ofthe plasmid carrying HS1 to give a plasmid carrying HSS 1 and 2 in thesame orientation as that found in the normal human β-globin locus,

3. A 1.5 kb Asp718-SalI fragment encompassing DNaseI HSS 3 was madeblunt-ended by treatment with Klenow fragment of DNA polymerasedeoxynucleotide triphosphates and cloned into the HindII site of GSE1365,

4. A 1.1 kb partial SacI fragment encompassing DNaseI HSS4 was similarlymade, blunt-ended and cloned into the SmaI site of the plasmid carryingHSS3 to give a plasmid with HSS3 and HSS4 in the same orientation asthat found in the normal human β-globin locus,

5. The 4.9 kb BglII fragment containing a normal copy of the humanβ-globin gene with 1.56 kb of 5' flanking sequence and 18 kb of 3'flanking sequence was cloned into the BamHI site of GSE 1366,

6. A 2.0 kb partial NarI fragment which includes the Herpes SimplexVirus (HSV) thymidine kinase (tk) promoter driving the expression of theTn5 neomycin resistance gene and 3' polyadenylated sequences of the HSVtk gene was cloned into the NarI site of the plasmid carrying the humanβ-globin gene and,

7. The four HSS, the human β-globin gene and the tkneo gene werecombined in a three fragment ligation using a PvuI-BstEII fragment ofHSS 1 and 2 with a BstEII ClaI fragment of HSS 3 and 4 and a ClaI-PvuIfragment of the plasmid containing the β-globin and tkneo genes. Thefinal construct was shown to direct high-level expression of the humanβ-globin gene in mouse erythroleukemic cells (MEL) independent of theposition of integration of the DNA in the host cell chromosome. Thelevel of β-globin expression was related to the gene copy number.

EXAMPLE 3

To test the effect of changing the position of the DNaseI HSS relativeto the β-globin gene, the DNaseI HSS fragments described in Example 2above were cloned so as to give a 6.5 kb NotI restriction fragment. ThisDNA fragment was cloned in both orientations into a unique NotI site intwo different plasmids containing the tkneo gene as a 2.7 kb partialEcoRI fragment and the human β-globin gene as a 4.9 kb BglII fragment asdescribed above.

The two tkneo β-globin plasmids differed in the orientation of theβ-globin gene and cloning of the 6.5 kb NST I fragment in bothorientations generated the four plasmids GSE 1359, GSE1400, GSE 1401 andGSE 1357 shown in FIG. 9.

The DNA of the four plasmids shown in FIG. 9 was linearized by digestionwith the restriction enzyme PvuI. The DNAs were recovered by ethanolprecipitation and introduced into MEL cells growing exponentially inalpha MEM medium supplemented with 10% fetal calf serum and antibiotics.

MEL cells (approx. 10⁷) were collected by centrifligation (1000 g for 10minutes) washed once with ESB Buffer (20 mM HEPES pH 7.0, 140 mM NaCl, 5mM KC1) and resuspended in 1 ml of ESB containing 100 μg of linearizedplasmid DNA. After incubation on ice for 10 minutes, cells were placedin a well of a 24 well tissue culture plate (Nunc) and a 10 ms shock wasdelivered using a Haeffer "Pro genitor" electroshock apparatus at avoltage gradient of 250 volts/cm (Antonious et al., EMBO J, (1988), 7,377-384).

Cells were recovered into 40 ml of αMEM and 10% fetal calf serum at 37°C. for 24 hours and G418 selection was applied by changing the tissueculture medium for αMEM with 10% fetal calf serum and 800 mg G418 (GibcoBRL) per ml. After 11 days in selection G418 resistant cell populationswere harvested by centrifugation. RNA and DNA was prepared and part ofthe culture was induced to undergo erythroid differentiation by growthin αMEM with 10% fetal calf serum and 2% dimethyl sulfoxide (DMSO-BDH).After four days, populations were harvested and RNA prepared byhomogenizing cells in 3M LiCl 6M urea.

After sonication and incubation at 4° C. overnight, RNA was precipitatedand analyzed by Northern blot analysis. RNA from uninduced (-) orinduced (+) MEL cell populations (10 mg) was denatured with 50%formamide 15% formaldehyde in 1 ×MOPS buffer heated at 60% for 10minutes, chilled on ice and loaded on to a 1% agarose gel. The gel wasrun at 70 volts/cm for 8 hours in 1 ×MOPS buffer (20 mM MOPS pH7, 5 mmsodium acetate, 20 mM EDTA). The gel was soaked in 20 ×SSC (3M NaCl 0.15trisodium citrate ) for 30 minutes then blotted directly to anitrocellulose filter (Schleicher and Schuller-0.1 μM) overnight. Thefilter was baked for 2 hours at 80° C. then hybridized to DNA probeslabeled to high specific activities by Nick translation (Rigby et al.,J. Mol. Biol. (1977) 113 237-251).

Hybridization was performed in buffer containing 50% formamide, 6×SSC,10×Denhardts 1% SDS and 200 μg salmon sperm DNA per ml (Lang et al.,EMOB J (1988), 7 (6), 1675-1682). After overnight hybridization, at 42°C., filters were washed to a final stringency of 0.1×SSC 0.1% SDS at 55°C. Filters were exposed to X-ray film with intensifying screens at 70°C. Specific DNA probes used in the experiments of FIG. 10 were:

Human β-globin

A 800 bp EcoRI-MspI fragment which includes the 3rd exon of the humanβ-globin gene.

Mouse α-globin

A 300 bp BamHI fragment of the mouse α-globin gene including sequencesfrom the 2nd exon.

tkneo

A 2.0 partial NarI fragment encompassing the tk promoter and the Neoresistance gene.

Mouse Histone H4 gene

A 300 bp HinfI fragment of a mouse histone H4 gene.

The experiment described above shows that:

1. Human β-globin RNA expression in induced MEL cells is as high orhigher than the expression of endogenous mouse α-globin genes.

2. β-globin expression using β micro locus constructs is higher thanthat obtained with a β mini locus in MEL cells,

3. The dominant activator sequences increase transcription from thetkneo gene approximately 100 fold compared to plasmids that do notcontain the dominant activator sequences and,

4. The dominant activator sequence works on the human β-globin gene inboth orientations upstream or downstream of the β-gene.

EXAMPLE 4

Experiments were conducted to identify any dominant activator sequencesin the human CD2 gene and to test their ability to direct integrationsite independent expression. Two DNaseI hypersensitive sites were found3' of the human CD2 gene which is expressed at a high level in thethymus oftransgenic mice. A 5.5 kb BamiHI -XbaI fragment of CD2 3'sequences including the DNaseI HSS was cloned next to a 4.8 kb BglIIhuman β-globin gene fragment (Example 5 below) or a 9.5 kb mouse/humanhybrid Thy-1 gene fragment. DNA fragments prepared from βCD2 and Thy-1CD2 constructs were introduced into fertilized mouse eggs. Transgenicmice with a range of copy numbers were obtained and analysis showedhigh-level expression of human β-globin (example 5) and mouse/humanhybrid Thy-1 gene mRNA in thymus (this example) which was related tocopy number and independent of the site of integration into the mousegenome.

The experiments described in this Example and in Example 5 demonstrateunequivocally the presence of a dominant activator sequence in the 5.5kb BamHI-XbaI fragment 3' of the human CD2 gene.

Mapping DNase HSS in the CD2 gene Transgenic mice carrying the 2.8 kbKpnI fragment of the human CD2 gene were sacrificed and nuclei preparedfrom thymus and liver of mouse lines CD2-4 and CD2-1 (Lang et al., EMBOJ, (1988), 7(6), 1675-1682). DNaseI digestions were performed asdescribed herein (see also Grosveld et al., Cell, (1988), 51, 975-985).

DNA samples were recut with the restriction enzymes indicated in FIG. 11and probed with 5' or 3' human CD2 probes. Strong DNase HSS were present5'of the gene and two 3' sites are seen 3' of the CD2 gene only intransgenic thymus not in liver nuclei.

FIG. 12 shows a Thy-1.1 CD2 construct. The stippled bar denoted humanThy-1 sequences and the two arrows show the position of the DNaseI HSSin the CD2 fragment.

A 5.5 BamHI-XbaI fragment 3' of the human CD2 gene shown to contain thetwo 3' DNase I HSS was cloned between the BamHI and XbaI sites of thebluescript vectors KS+(Promega). A 9.5 kb EcoRI fragment containing amouse-human hybrid gene (Kollias et al., Cell, (1987), 51, 21-23), wascloned into the EcoRI site next to the 5.5 kb BamHI-XbaI fragment togive the plasmid Thy-1 CD2-A shown in FIG. 12. Plasmid sequences wereremoved by digestion with restriction endonucleases SalI and NotI andthe 15 kb Thy-1 CD2 insert was purified by gel electrophoresis andpassage over an Elutip D column (Schleicher and Schuell).

The purified DNA was introduced into the nuclei of fertilized CBA ×C57BL-10 mouse eggs by microinjection (Lang et al., EMBO J (1988), 7,1675-1682). Injected eggs were transplanted into the oviduct of pseudopregnant foster mothers by standard techniques (Hogan et al.,"Manipulating the mouse embryo" Cold Spring Harbor publications (1986)).Transgenic mice were identified by Southern blot analysis of tail DNA asdescribed previously (Lang et al., Loc. cit.) and tissues were dissectedfor RNA preparation when animals were 3 to 4 weeks old.

Referring to FIG. 13 RNA from thymus or brain of transgenic mice1,2,3,5,6,7 and 8 (5 μg) was hybridized with a 5' end labeled TthIII -SacI mouse Thy-1 IVth exon probe and a 3' end labeled BglII Neo humanThy-1 probe at 52° C. for 12 hours after melting the probes or RNA at85° C. for 15 minutes.

RNA hybrids were digested with Nuclease S1 (Boehringer Mannheim) asdescribed previously (Kollias et al., Cell, (1988), 51, 21-31) andprotected fragments run out on a 5% polyacrylamide 8M urea sequencinggel. The positions of protected fragments for endogenous mouse andtransgenic mouse human Thy-1 RNAs are indicated.

The level of expression per gene copy of the hybrid mouse human Thy-1gene in transgenic thymus is as high as that for the endogenous Thy-1gene and is related to the gene copy number (mouse 1 has approximately20 copies of the transgene; mouse 2 has 10 copies of the transgene).

EXAMPLE 5

This example refers to FIG. 14 which shows the construction of aβ-globin CD2 plasmid.

β-globin CD2 expression in transgenic mice was brought about usingdominant activator sequences of the invention.

The inserts of plasmid β CD2-A and B-CD2-β were isolated by agarose gelelectrophoresis after digestion with restriction enzymes Nat I and SalI. DNA was injected into nuclei of fertilized mouse eggs as describedabove (see also Grosveld et al., Cell (1987), 51, 975-985.) Transgenicmice were identified by Southern blot analysis of tail DNA using a humanβ-globin indicator probe (900 bp BamHI-EcoRI fragment).

FIG. 15 shows Smg of genomic DNA from transgenic mice carrying eitherthe β CD2-A or β CD2-βconstruct applied to a nitrocellulose filter usinga slot blot manifold (Schleicher and Scheull). The filter was hybridizedto a nick-translated β-globin 2nd intron probe and washed to a finalstringency of 0.1×SSC 0.1% SDS at 65° C. The strength of thehybridization signal is a direct measure of the number of gene copies oneach mouse.

FIG. 15B shows an S1 Nuclease analysis of thymus RNA prepared fromtransgenic mice. RNA (1 mg) was hybridized with a 5' end labeled 52.5 bpAccI fragment of β-globin cDNA construct (Grosveld et al., Cell, (1987),51, 975-985) and a 300 bp Hinf I fragment of a mouse histone H4 gene asan internal control.

After hybridization at 52° C. for 12 hours, samples were digested withNuclease S1 and protected fragments analyzed on a 5% polyacrylomide 8Murea gel. The positions have protected fragments for endogenous histoneH4 and human β-globin mRNA are indicated. The intensity of the humanβ-globin-protected fragment is a direct measure of the amount of humanβ-globin mRNA transcribed in the thymus. For comparison 0.1 mg of fetalliver RNA from a transgenic mouse carrying 4 copies of the β-micro locuswas used in the track labeled 33 FL.

Referring to FIG. 14, the 5.5 kb BamHI and XbaI fragment of human CD2gene was cloned between the BamHI and XbaI sites of the bluescriptvector KS+(Promega). The 4.9 kb BglII fragment of the human β-globingene was cloned into the BamHI site of this plasmid in both orientations(β CD2A and β CD2-β). The vertical arrows indicate positions of CD2DNaseI HSS. The black bars indicate exons of the human β-globin gene andhorizontal arrows show the orientation of the gene fragments used.

It will be understood that the invention is described by way of exampleonly and modifications may be made within the scope of the invention.

EXPERIENCES

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Angel, P., Imagawa, M., Chin, R., Stein, B., Imbra, R., Rahmsdorf, M.,Jonat, A., Herrlich, P. and Karin, M. (1987) Phorbol ester-induciblegenes contain a common cis element recognized by a TPA-mediatedtransacting factor. Cell 49, 729-739.

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We claim:
 1. An isolated DNA molecule, comprising:(i) a dominantactivator sequence that is specific for a particular mammaliancell-type, and (ii) a structural gene, whereinthe region in said DNAconsisting of (i) and (ii) and DNA therebetween has a nucleotidesequence different from that of naturally occurring DNA, and saiddominant activator sequence being characterized in that, in naturallyoccurring DNA:(I) it is associated with a naturally occurring gene thatis expressed in a tissue-specific manner; and (II) it is locatable innaturally occurring DNA by association with a DNase I superhypersensitive site; wherein said dominant activator sequence ischaracterized in that it stimulates expression of said structural genewhen said DNA molecule is integrated into a genome of a host cell ofsaid mammalian cell-type, such that said expression:(a) is dependent onthe number of copies of said gene that are integrated into said genomein that said expression increases as said number of copies of said geneincreases; and (b) is independent of the integration site of said DNAmolecule in said genome.
 2. A DNA molecule according to claim 1, whereinsaid dominant activator sequence is a dominant activator sequence fromthe human β-globin locus.
 3. A DNA molecule according to claim 2,wherein said dominant activator sequence is a dominant activatorsequence of the 21 kb fragment of human genomic DNA extending from theClaI site 1 kb 5' to the epsilon-globin gene in the β-globin locus tothe BglII site 22 kb 5' to the epsilon-globin gene.
 4. A DNA moleculeaccording to claim 3, wherein said dominant activator sequence comprisesone or more of the following fragments of said 21 kb fragment of humangenomic DNA:the 2.1 kb XbaI-XbaI fragment, the 1.5 kb KpnI-BglIIfragment, and the 1.1 kb SacI-SacI fragment which comprises an internalSacI site.
 5. A DNA molecule according to claim 4, wherein said dominantactivator sequence further comprises the 1.9 kb HindIII-HindIII fragmentof said 21 kb fragment of human genomic DNA.
 6. A DNA molecule accordingto claim 1, wherein said dominant activator sequence is a dominantactivator sequence from the human CD2 locus.
 7. A DNA molecule accordingto claim 6, wherein said dominant activator sequence is within the 5.5kb BamHI-XbaI fragment of human genomic DNA immediately 3' to the CD2gene.
 8. A mammalian host cell transformed with a DNA molecule accordingto claim
 11. 9. A method of producing a polypeptide, comprisingculturing a mammalian host cell transformed with a DNA moleculeaccording to claim
 1. 10. The method of claim 9, wherein said dominantactivator sequence is a dominant activator sequence from the humanβ-globin locus.
 11. The method of claim 10, wherein said dominantactivator sequence is a dominant activator sequence of the 21 kbfragment of human genomic DNA extending from the ClaI site 1 kb 5' tothe epsilon-globin gene in the β-globin locus to the BglII site 22 kb 5'to the epsilon-globin gene.
 12. The method of claim 11, wherein saiddominant activator sequence comprises one or more of the followingfragments of said 21 kb fragment of human genomic DNA:the 2.1 kbXbaI-XbaI fragment, the 1.5 kb KpnI-BglII fragment, and the 1.1 kbSacI-SacI fragment which comprises an internal SacI site.
 13. The methodof claim 12, wherein said dominant activator sequence further comprisesthe 1.9 kb HindIII-HindIII fragment of said 21 kb fragment of humangenomic DNA.
 14. The method of claim 9, wherein said dominant activatorsequence is a dominant activator sequence from the human CD2 locus. 15.The method of claim 14, wherein said dominant activator sequence iswithin the 5.5 kb BamHI-XbaI fragment of human genomic DNA immediately3' to the CD2 gene.
 16. An isolated DNA molecule, comprising:(i) adominant activator sequence that is specific for a particular mammaliancell-type, (ii) a promoter that is funtional in said particularmammalian cell-type, and (iii) a structural gene operatively linked tosaid promoter, whereinthe region in said DNA consisting of (i), (ii) and(iii) and DNA therebetween is constructed so as to have a nucleotidesequence different from that of naturally occurring DNA, and saiddominant activator sequence being characterized in that, in naturallyoccurring DNA:(I) it is associated with a naturally occurring gene thatis expressed in a tissue-specific manner; and (II) it is locatable innaturally occurring DNA by association with a DNase I superhypersensitive site; wherein said dominant activator sequence ischaracterized in that it stimulates expression of said structural genewhen said DNA molecule is integrated into a genome of a host cell ofsaid mammalian cell-type, such that said expression:(a) is dependent onthe number of copies of said gene that are integrated into said genomein that said expression increases as said number of copies of said geneincreases; and (b) is independent of the integration site of said DNAmolecule in said genome.